The neuronal differentiation of PC12 cells is being further defined in three ways: (1) the in vitro effect of brain cells on the neuronal differentiation of PC12 cells, treated with nerve growth factor (NGF) or infected with ras~oncogene, that might account for the survival of PC~12 cells grafted to brain. Specifically ,the expression of choline acetyl transferase (ChAT) and acetylcholine esterase (AChE) are assayed biochemically. Structural changes are assessed by electron microscopy. PC12 cells, differentiated either by NGF or ras~oncogene, are cocultured with astrocytes or brain endothelial cells. After 6~14 daysin coculture, the number of neurites, their varicosities and clusters of synaptic vesicles, identified immunocytochemically by their content of synapsin and synaptophysin, all increase over PC12 cells in solo culture. As controls, fibroblasts were used instead of astroglia and bovine aortic endothelium instead of brain endothelium. The release of 32P~dopamine after K+ stimulation is 3~5 times greater from ras~PC12 cells than from naive cells in solo culture. (2) As a major component of cell surface adhesion molecules that may affect the association of PC12 cells with brain cells, sialic acid, measured by HPLC, decreases after NGF treatment but rises markedly in oncogene treated PC~12 cells. (3) The signaling pathway for the expression of growth~associated protein (GAP)~43 induced by NGF and by ras~oncogene are being compared. The time course for the expression of GAP~43 and the augmented neurite outgrowth were similar in cells treated with NGF or with ras~oncogene. Both events appear to be signaled through a pathway involving activation of the regulatory G protein, p21, encoded by the oncogene.